Insect ecdysis sequences represent a simple, robust, and tractable model for studying the neuromodulatory mechanisms that govern behavior. Because initiation of an ecdysis sequence involves a profound shift in behavioral priorities, study of these sequences offers the opportunity to understand the neuromodulatory mechanisms that govern changes in behavioral state. In addition, because ecdysis behaviors are inherently sequential, they permit the systematic investigation of how motor programs are assembled and serially executed by the nervous system. Finally, the study of ecdysis sequences promises insight into how conserved circuits can be variably configured to generate immensely different behaviors. In Drosophila, for example, the motor sequences performed at pupal and adult ecdysis before and after metamorphosis, respectively are scarcely similar though they are governed by a common set of neuromodulatory/hormonal inputs. By analogy to computing, these inputs can be regarded as instructions written in a higher programming language that are then compiled into different motor output patterns. Exposing the mechanisms of neural compilation in ecdysis is likely to deeply inform our understanding of how neuromodulators contribute to neurocomputation by reconfiguring the activity of neural networks. To investigate these questions, our laboratory seeks to elucidate the circuitry that governs both the pupal and adult ecdysis sequences in Drosophila, with our primary efforts over the last year being devoted to the pupal ecdysis circuitry. As noted above, pupal ecdysis is strictly dependent on intrinsic factors, a fact most evident from the observation that exogenous application of the peripherally released hormone, Ecdysis Triggering Hormone (ETH) to an isolated pupal nervous system is sufficient to trigger sequential activation of neurons that express other key neuromodulators governing ecdysis. Evidence indicates that this sequential activation corresponds to the generation of a fictive ecdysis sequence, and our efforts over the last year have focused on characterizing the neuronal substrates of this fictive sequence using functional manipulations and Ca++ imaging. Following up on our previous identification of neurons directly targeted by ETH (Diao et al., 2016, Genetics 202:175-189), we are now characterizing neurons that act downstream of these targets. Of principal interest are neurons that respond to two hormones known to play important roles in generating the pupal ecdysis sequence: Bursicon and CCAP. These hormones are co-released by a small subset of ETH targets after the initiation of pupal ecdysis and have been shown to initiate the second of three phases of the pupal ecdysis sequence. We have completed an initial characterization of the neuronal populations that respond to Bursicon and CCAP, and this work is the subject of a recently submitted manuscript. It is worth noting that this work relied heavily on two genetic targeting methods previously developed in the laboratory: The Split Gal4 system for refined transgene targeting (Luan et al., 2006, Neuron 52, 425-436) and the Trojan exon method (Diao et al., 2015, Cell Reports 10, 1410-1421). Our success in applying these techniques to elucidating the pupal ecdysis circuit illustrates a general strategy for mapping hormonally regulated networks: Using genes encoding the receptors of behaviorally relevant hormones as entry points for refined genetic and neuronal manipulations, one can establish patterns of functional connectivity between neurons in a behavioral network. The Split Gal4 system also formed the basis of two papers published in collaboration during the past year. The first describes the Split Gal4-facillitated dissection of a neural circuit that coordinates male copulation behavior in Drosophila (Pavlou et al. eLife 2016; 5:e20713. DOI: 10.7554/eLife.20713). The second describes the development of a novel genetic tool called the Killer Zipper, which augments the targeting capabilities of the Split Gal4 system (Dolan et al., Genetics 206, 775-784). The Killer Zipper is a suppressor of Split Gal4 activity that permits further refinement of the already restricted neuronal targeting that can be achieved with the Split Gal4 system. We introduce in the paper a versatile toolkit of Killer Zipper constructs and fly lines that should generally facilitate neural circuit mapping efforts in the fly and is already helping spur our own on-going investigation of the circuitry underlying the adult ecdysis sequence. In summary, we have made good progress during the last year in elucidating the functional architecture of the neural network that underlies execution of the pupal ecdysis sequence. At the same time, we have continued to develop tools that will support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the circuitry underlying ecdysis sequences at all developmental stages in the fly, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.